Stimulation of Proliferation of a Human Osteosarcoma Cell Line by Exogenous Acidic Fibroblast Growth Factor Requires both Activation of Receptor Tyrosine Kinase and Growth Factor Internalization

Abstract

U2OSDr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell.Acidic fibroblast growth factor (aFGF) induces a multitude of responses in target cells. In many cells, this growth factor is a potent mitogen (3, 9, 23, 24); in others, it inhibits proliferation (34) or induces various kinds of differentiation (6,17,27,39,41,44,50,58). The different responses may partly be due to the fact that there are four known FGF receptor genes and a number of splicing variants for three of them (11,22,26,64,65).In a previous paper, we showed by cell fractionation that externally added aFGF was recovered in the nuclear fraction of NIH 3T3 cells, which contain specific FGF receptors. We also presented evidence that targeting to this location is required for the stimulation of DNA synthesis, whereas external binding to the receptor is sufficient to induce tyrosine phosphorylation (62). In Vero Dr22 and U2OS Dr1 cells, which lack specific FGF receptors and do not respond to aFGF, we were able to stimulate DNA synthesis by translocating the growth factor into the cytosol as a fusion protein with the diphtheria toxin A fragment (aFGF-dtA). Reconstituted with the diphtheria toxin B fragment (dtB), the fusion protein was translocated by the diphtheria toxin pathway (42, 43) into the cytosol of Vero Dr22 and U2OS Dr1 cells, which are insensitive to the intracellular action of diphtheria toxin but rich in diphtheria toxin receptors, and it was subsequently found in the nuclear fraction (63). However, there was no stimulation of cell proliferation.In NIH 3T3 ...

FGF-3 and FGF-4 elicit distinct oncogenic properties in mouse mammary myoepithelial cells

“…It thus follows that FGF-3 and FGF-4 expressed in the same cell type and from identical constructs, have distinct modes of action and confer to the EF43 cells an invasive, tumorigenic and metastatic phenotype via signaling pathways that lead to transcriptional activation of distinct genes. Because translocation within the cell has been demonstrated for FGF-1 and 2 (Wiedlocha et al, 1996), an FGF intracellular signal cannot be excluded as a possible mechanism of action. However, FGF-4 transforms cells through an autocrine loop that involves the extracellular activation of the receptors (Talarico and Basilico, 1991).…”

Inability of the Acidic Fibroblast Growth Factor Mutant K132E to Stimulate DNA Synthesis after Translocation into Cells

“…To check that the mutated aFGF had retained its ability to bind to FGF receptors, we added the labeled growth factor to different cell lines and kept them on ice for 2 h, then the cells were washed and analyzed by SDS-PAGE. In the absence of heparin, wild-type growth factor bound extensively to untransfected U2OSDR1 cells as well as to the same cells transfected with FGF receptor 4 (U2OSDR1-R4) (12) and to NIH 3T3 cells expressing FGF receptor 1 (22) (Fig. 1A, lanes 1, 4, and 7), while aFGF(K132E) bound to a lesser extent under the same conditions (lanes 10 and 16).…”

“…aFGF(K132E) Is Translocated to the Nuclear FractionThere are several reports that externally added aFGF is translocated to the nuclear fraction in cells containing FGF receptors (4,5,12,23,24). This transport appears to be required for stimulation of DNA synthesis, at least in certain cells (3,4,12).…”

“…Recombinant proteins were expressed and purified as described previously (12) except that for aFGF(K132E) and aFGF(K132E)-CAAX the clarified bacterial lysates were applied to heparin-Sepharose in a buffer containing 0.1 M NaCl instead of 0.5 M and were further purified using an Econo-Pac Q cartridge (Bio-Rad).…”

Endogenous basic fibroblast growth factor isoforms involved in different intracellular protein complexes

“…On the other hand, the nuclear CUG-initiated forms are involved in cell proliferation by a cell-surface receptorindependent pathway [13]. Concurrently with bFGF, the intracellular route has been recently analysed for acidic FGF [15,16], and the results strongly suggest two distinct but complementary endogenous and exogenous pathways [17].…”